We have obtained evidence indicating that the unconjugated N-hydroxylated derivatives of the bladder carcinogens 4-biphenylamine, 1-naphthylamine, and 2-naphthylamine play a key role in the etiology of bladder cancer in the dog. Further, we have shown that the source of the unconjugated N-arylhydroxylamine metabolite of 4-biphenylamine in the bladder of the dog is derived from the non-enzymatic and/or enzymatic hydrolysis of a very labile O-glucuronide ester formed in the liver for renal excretion. We propose to determine if the source of the unconjugated N-arylhydroxylamines of a series of aromatic amines known to induce bladder cancer in the dog are also derived from their O-glucuronide esters. The chemical synthesis of these esters will be undertaken. It remains to be established if the unconjugated N-arylhydroxylamines or their O-glucuronide esters are the ultimate carcinogens (triggering the carcinogenic response in the target tissue) or whether they require a further transformation (e.g., oxidation to the nitroso derivatives). A comparative study of the type and extent of the interaction of a series of carcinogenic N-arylhydroxylamines, their O-glucuronide esters, and nitroso derivatives with selected cellular macromolecules and constituents isolated from dog bladder mucosa, both in vivo and in vitro, will be undertaken. We propose to investigate the possibility of the application of immunochemical techniques for 1) the detection of 4-biphenylamine and its nitroso and N-hydroxy derivatives, and 2) for the detection of modified bases in nucleic acids produced by interaction with selected carcinogenic N-oxidation products.